Autoimmune diseases are often attributed to activation of self-reactive T cells specific for a restricted epitope on a self antigen. To date, treatments of autoimmune diseases have focused predominantly on nonspecific immunosuppressive approaches rather than focusing directly on the autoreactive T cell population. In these disease states, it would be useful to be able to induce T cell tolerance towards known antigens. T cells recognize peptide antigen bound in the groove of MHC molecules by means of the T cell receptor (TCR). Variable regions on both the a and b chains (Va and Vb) are involved in the binding of the MHC/peptide complex. However, there exists a class of antigens called superantigens (SAG) that binds a different region of MHC class II molecules and activates T cells based solely on the family of Vb that the TCR utilizes. Superantigens can be encoded endogenously by mouse mammary tumor viruses (mtv) in the mouse genome, or are synthesized exogenously by some bacteria and viruses. Encounter of these superantigens in the peripheral immune system leads to T cell activation first, followed by tolerance induction resulting from apoptosis. What dictates whether an antigen will activate or tolerize a T cell is not fully understood. We have initiated experiments to create a hybrid superantigen that maintains the tolerance-inducing activity of the superantigen yet can be directed towards T cells of any desired specificity. The initial construct was created by joining the nonpolymorphic region of the gene encoding the mtv 1 SAG, which encodes the MHC-binding domain, to a nucleotide sequence encoding a peptide antigen, 88-104 of pigeon cytochrome C. A control construct with the native sequence of the mtv 1 SAG has also been generated. These constructs will be expressed in a baculovirus expression system, purified using a histidine tag, then characterized for the ability to bind MHC molecules. Once binding to MHC has been established, the ability of this recombinant moleculte to be recognized by cytochrome C-specific T cells will be assessed in vitro by analysis of T cell proliferation, cytokine release, and induction of apoptosis. Studies will be performed to address the fate of T cells in vivo that recognize this superantigen hybrid. Because there is generally a very low precursor frequency of T cells specific for any one antigen, mice expressing a transgenic T cell receptor specific for the cytochrome C peptide will be used in which all of the T cells express the same TCR. Should successful tolerance induction be observed, these experiments will be extended to mouse models of autoimmune disease in which the construct will be altered to express the epitope of a self peptide involved in the disease, in attempts to determine whether this novel molecule will be useful therapeutically for the treatment of autoimmunity.